There is a need, in the field of biomaterial implantation, for resorbable and swellable particles charged with fragile bioactive drugs such as macromolecules. However, only incomplete solutions have been devised thus far.
Macromolecules are new types of molecules having particularly interesting therapeutical uses. Especially the specialized biological activities of these types of drugs provide tremendous advantages over other types of pharmaceutics. Examples of macromolecules are proteins and nucleic acids.
Currently more than 130 proteins are marketed (Leader, 21-39, 2008, Nature Reviews). More and more protein drugs are being employed in clinical trials because of advances in biotechnology allowing mass production of recombinant proteins. They are used to treat patients suffering from numerous diseases: cancer (treatment with monoclonal antibodies and interferons), cardiovascular disease, cystic fibrosis, Gaucher disease (treatment with enzymes and proteins in the blood), diabetes (insulin), anemia (erythropoietin), bone defects (bone morphogenetic proteins) and hemophilia (coagulation factors).
Nucleic acids are also macromolecules with medical applications. These therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. These drugs can be used to mitigate disease either prophylactically or at a very early stage, preventing disease progression and its complications
This type of molecules is very fragile and contact with organic solvent, use of high temperature, shear stress or acidic environment should be avoided. Therefore delivery of macromolecules in the body is a challenge.
Indeed, due to their nature, macromolecules cannot be administered orally. These products tend to degrade rapidly in the gastrointestinal tract, in particular because of the acidic environment and the presence of enzymes therein. Furthermore macromolecules have short in vivo lives.
Moreover, to a higher extent macromolecules are not able to pass endothelial, epithelial and intestinal barriers, due to their size and, generally, polar character.
For these reasons, macromolecules have to be brought in the system parenterally, i.e. by injection. The pharmacokinetic profile of these products is, however, such that injection of the product requires a frequent administration. Sometimes even multiple daily injections or continuous infusions are required for the protein drug to have a desired therapeutic effect. It will be evident that this is inconvenient for patients requiring these drugs. Furthermore, this type of application often requires hospitalization and/or medical supervision and has logistic drawbacks.
In addition, it appears that at least for certain classes of pharmaceutical macromolecules, such as cytokines which are presently used in e.g. cancer treatments, the therapeutic efficacy is strongly dependent on effective delivery on the site where it is needed. In such cases, the macromolecules should be directed to the sites where their activity is needed during a prolonged period of time.
Hence, there is a need for delivery systems which have the capacity for controlled release. In the art, delivery systems consisting of polymeric networks in which the macromolecules such as proteins are loaded and from which they are gradually released have been proposed.
The local delivery of macromolecules is challenging. The main drawback of this approach is the requirement of a polymer solubilization step using a solvent, such as methylene chloride or isopropanol, high temperature, foaming, which can compromise macromolecule stability. Local delivery of nucleic acid into microparticles has several limitations, similar to proteins. These problems include damage to DNA during microencapsulation, low encapsulation efficacy and minimal initial release of entrapped compound (O'Hagan, 10-19, 2006, Methods).
One of the most important differences affecting delivery and biological effectiveness of macromolecules in particular protein is complexity of protein structure, close relation between protein efficacy and molecular three-dimensional structure. It is essential to maintain the structural integrity through all the formulations steps of local drug delivery system. The method of encapsulation of proteins in a system of delivery is a critical step that can lead to inactivation of the protein (Sinha and Trehan, 261-280, 2003, Journal of Controlled Release). The most commonly used methods for protein drug encapsulation in polymeric microparticles include solvent extraction or evaporation from a W1/O/W2 dispersion, coacervation, and spray drying (Dai, 117-120, 2005, Colloids and Surface B, Freitas, 313-332, 2005, Journal of Controlled Release; Sinha and Trehan, 261-280, 2003, Journal of Controlled Release; Tamber, 357-376, 2005, Advanced Drug Delivery Reviews). Many disadvantages are associated with these methods which could cause protein denaturation and instability during encapsulation and release process. Double emulsion method has the limitations of exposure to organic solvents, high shear stress, and aqueous organic interfaces. Spray drying operates at elevated temperature and is not advisable to be used for highly temperature sensitive compounds such therapeutic proteins and nucleic acids.
Briefly, proteins have four levels of structural organization: the primary structure of the linear chain of amino acids along the polypeptide chain, the secondary structure formed by the local folding of the amino acids in a region of polypeptide chain in helices or sheets, the tertiary structure made of a stable arrangement in space of the helices and sheets. The quaternary structure is an arrangement of subunits of these proteins, the active protein consists of several subunits, such as hemoglobin or antibodies (assembly of four protein chains by disulfide bridges established between 2 cysteines). The tertiary or quaternary structure of proteins depends on weak bonds (hydrogen, hydrophobic, ionic) established between amino acid residues of polypeptide chains. These non-covalent bonds are fragile and can be broken under certain conditions leading to protein unfolding. The biological function of a protein depends of its structure, denatured protein can no longer perform its function. The main factors causing protein denaturation are the heat that breaks the weak hydrogen bonds, the pH (too acidic or too alkaline) and the ionic strength. A protein unfolding also occurs in presence of organic solvent, where the hydrophobic regions of folded proteins are turning to outside while the hydrophilic regions will gather in the center of the molecule. During the preparation of microspheres, the proteins can be exposed to both high temperature and organic solvents such as dichloromethane (solvent of PLGA) and be denatured (Raghuvanshi, 269-276, 1998, Pharm Dev Technol).
More in detail, at present, two major types of polymeric delivery systems can be distinguished: biodegradable polymers and non-biodegradable hydrogels.
Biodegradable polymers, e.g. polylactic acid (PLA) and copolymers of PLA with glycolic acid (PLGA), are frequently used as delivery systems for macromolecules such as proteins.
Macromolecules can be incorporated in pharmaceutical delivery systems, e.g. microspheres, by a variety of processes. In vitro and in vivo, usually a biphasic release profile is observed: an initial burst followed by a more gradual release. The burst is caused by macromolecules present at or near the surface of the microspheres and by macromolecules present in pores. The gradual release is ascribed to a combination of diffusion of the macromolecules through the matrix and degradation of the matrix. Especially for larger macromolecules diffusion in these matrices is negligible, so that the release depends on the degradation of the polymer. The degradation can be influenced by the (co)polymer composition. A well-known strategy to increase the degradation rate of PLA is co-polymerization with glycolic acid.
Although delivery systems based on biodegradable polymers are interesting, it is very difficult to control the release of the incorporated macromolecule. This hampers the applicability of these systems, especially for macromolecules with a narrow therapeutic window, such as cytokines and hormones. Furthermore, organic solvents have to be used for the encapsulation of the macromolecules in these polymeric systems. Exposure of macromolecules to organic solvents generally leads to denaturation, which will affect the biological activity of the macromolecules. Furthermore, the very stringent requirements of registration authorities with respect to possible traces of harmful substances may prohibit the use of such formulations of therapeutic drugs in human patients.
Moreover PLGA hydrolysis in the core of microspheres causes a local acidification. The pH was measured using pH-sensitive organic probes and values between 1.5-3 were obtained (Fu, 100-106, 2000, Pharmaceutical Research; Li and Schwendeman, 163-173, 2005, Journal of Controlled Release). The pH drop within polymer matrix of microspheres can induced different alterations of proteins encapsulated in PLGA matrix. The more documented adverse reactions affecting encapsulated proteins are deamidation, acylation and hydrolysis of the peptide bond (Murty, 50-61, 2005, International Journal of Pharmaceutics; Abbas Ibrahim, 241-252, 2005, Journal of Controlled Release, Houchin and Topp, 2395-2404, 2008, Journal of Pharmaceutical Sciences). The deamidation of proteins is an acid-catalyzed reaction in which the amino acids asparagine and glutamine are degraded to aspartic acid and glutamic acid. A significant deamidation was observed for encapsulated insulin (Uchida, 234-236, 1996, Chem Pharm Bull, Shao and Bailey, 623-632, 1999, Pharm Dev Technol). Acylation is another alteration of proteins entrapped within PLGA microspheres. During microspheres resorption, the proteins can be acylated with glycolic acid or lactic acid adducts. This side reaction was observed in vivo for the Octreotide peptide subcutaneously implanted in PLGA microparticles (Murty, 50-61, 2005, International Journal of Pharmaceutics) and in vitro for salmon calcitonin (Lucke, 175-181, 2002, Pharmaceutical Research). Acylation occurs on several amino acids: free amine of N-terminal amino-acid of protein, lysine, tyrosine or serine located along the peptide chain. The local acidity within the microspheres can cause hydrolysis of the peptide chain, particularly at the level of aspartic acid, the Asp-Pro bond is considered fragile. During in vitro release experiment, more than 50% of carbonic anhydrase released after one week from PLGA microspheres (1-3 microns) corresponding to fragments (Sandor, 63-74, 2002, Biochimica and Biophysica Acta).
Loaded PLGA microspheres were themselves loaded onto an alginate hydrogel to control the release for prolonged time periods (Lee, Journal of Controlled Release 137, 196-202, 2009). The bioactive compound is still incorporated in PLGA microsphere which may denature its structure during degradation.
Also hydrogels are frequently used as delivery systems for proteins and peptides. Hydrogels can be obtained by crosslinking a water-soluble polymer yielding a three-dimensional network which can contain large amounts of water. Proteins can be loaded into the gel by adding the protein to the polymer before the crosslinking reaction is carried out or by soaking a preformed hydrogel in a protein solution. So, no (aggressive) organic solvents have to be used to load the hydrogels with protein molecules.
In contrast to the biodegradable polymers, the release of proteins from hydrogels can be easily controlled and manipulated by varying the hydrogel characteristics, such as the water content and the crosslink density of the gel. However, a major disadvantage of the currently used hydrogel delivery systems is that they are not biodegradable. This necessitates surgical removal of the gel from the patient after the release of the protein in order to prevent complications of inclusion of the empty hydrogel material (wound tissue is frequently formed).
Biodegradable hydrogels have been used in the preparation of delivery systems for protein drugs. One of these systems comprises crosslinked dextrans obtained by coupling glycidyl methacrylate (GMA) to dextran, followed by radical polymerization of an aqueous solution of GMA-derivatizad dextran (dex-GMA) (Hennink, Pharmaceutical Research, vol 15, no 4, 1998). But size of the microspheres obtained is quite small (around 100 μm).
Proteins can be encapsulated in the hydrogels by adding proteins to a solution of GMA-derivatized dextran prior to the crosslinking reaction. It appeared that the release of the proteins out of these hydrogels depends on and can be controlled by the degree of crosslinking and the water content of the gel (Hennink et al., J. Contr. Rel. 39, (1996), 47-57).
Although the described cross-linked dextran hydrogels were expected to be biodegradable, these hydrogels are rather stable under physiological conditions.
To solve this problem US2008/131512 proposes to add a synthetic resorbable polymer (PLA or PGA) between dextran and the methacrylic double bond to facilitate the degradation of the network by hydrolysis. However, due to its chemical nature, dextran is quite difficult to modify in order to introduce specific functional groups onto the structure. These functional groups may help to favour the incorporation of the macromolecules and/or to control the release.
Microspheres can be made with natural polymer (gelatine or collagen). However, the release is rapid (in one week after implantation in rabbit joint cavity) (Inoue, 264-270, 2006, Arthritis & Rheumatism) and precluding their used for long-term and controlled drug delivery. It should also be noted that collagen is of bovine origin and allergic reactions to the bovine proteins are noted in about 2% of patients.
It is therefore a goal of the present invention to solve the above problems, in particular to avoid the macromolecule, such as protein, instability during microspheres preparation, the nonspecific interactions with the polymers, local acidity within the degrading polymer matrix and to obtain a better predictability and control of release of the microsphere with a limited burst effect.